Cetuximab is a human–murine chimeric monoclonal antibody to the epidermal growth factor receptor, active for advanced colorectal cancer treatment in combination with chemotherapy. Cetuximab mainly acts by inhibiting epidermal growth factor receptor-mediated pathways in cancer cells; however, in the human host, its IgG1 backbone may offer additional antitumor activity that includes FcγRs-mediated antibody-dependent cell cytotoxicity, phagocytosis, cross priming, and tumor-specific T-cell–mediated immune response. These mechanisms are still under active investigation. At this purpose, we have performed an immunologic investigation in advanced colon cancer patients enrolled in an ongoing phase II trial aimed to test the toxicity and the biological and antitumor activity of a novel biochemotherapy regimen combining polychemotherapy with gemcitabine, irinotecan, levofolinic acid, and fluorouracil with cetuximab and with subcutaneous low-dose metronomic aldesleukin (GILFICet regimen). The peripheral blood mononuclear cells of the first 20 patients enrolled in the GILFICet trial were collected at baseline and after 6 treatment cycles and examined for immune-phenotype change by flow cytometry. Colon cancer-specific T-cell lines were also generated ex vivo from these samples and subsequently characterized for immune phenotype, functional activity, and antigen specificity. We found a treatment-related increase of circulating dendritic cells, natural killer cells, central memory T cells, and activated T cells with a T-helper 1 (Th1)-cytotoxic phenotype. In addition, the ex-vivo characterization of antigen-specific T cells derived from the treated patients revealed a significant increase in proliferating cytotoxic T-lymphocyte precursors specific for carcinoembryonic antigen and thymidylate synthase derivative epitope peptides. On these basis, we concluded that the GILFICet regimen exerts substantial immune-modulating activity that significantly affects tumor antigen-specific T-cell compartment with potential antitumor activity.